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Genetics > Lecture--Chapter 19 > Flashcards

Lecture--Chapter 19 Flashcards

1
Q

use molecular techniques to produce new combinations of DNA fragments

A

recombinant DNA technology

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2
Q

What are the aspects of recombinant DNA technology?

A
  1. Disease diagnostics and gene therapy
  2. Make recombinant vaccines
  3. Express proteins in cells for therapeutic products (human insulin)
  4. Make transgenic organisms with value added traits
  5. Clone ORFs and regulatory regions for study
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3
Q

methods used to assemble recombinant DNA molecules and direct their replication within host organisms

A

gene cloning

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4
Q

Gene cloning involves both ____ DNA and ____ DNA.

A

chromosomal; vector

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5
Q

a DNA molecule used to transfer foreign DNA into another cell, where it can be replicated and/or expressed

A

vector DNA

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6
Q

small, circular, double-stranded DNA molecules in bacteria that carry genes which benefit survival of the bacterium

A

plasmids

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7
Q

What are the important features of plasmids?

A
  1. Strong origins of replication
  2. Multiple cloning sites
  3. A selectable marker
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8
Q

Important features of plasmids: Strong origins of replication:

A

enable their expression in high copy numbers

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9
Q

Important features of plasmids: Multiple cloning sites:

A

enable using different restriction enzymes to digest the DNA

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10
Q

Important features of plasmids: A selectable marker:

A

is a gene that codes for a product (e.g. antibiotic) that allows only transformed bacteria to survive

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11
Q

enzymes that cut DNA at or near specific recognition nucleotide sequences known as restriction sites

A

restriction enzymes (restriction endonuclease)

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12
Q

What are the aspects of Type II RE’s?

A
  1. recognition sites that are usually undivided, palindromic, 4-8 nt long
  2. recognise and cleave DNA at the same site, require Mg2+ as a cofactor
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13
Q

What are the steps of production of rDNA?

A
  1. Incubate both DNAs and EcoRI, which cuts the DNA backbone between G and A.
  2. Incubate the DNAs together, allowing sticky ends to hydrogen bond.
  3. Add DNA ligase, which covalently links the DNA backbones.
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14
Q

Steps for gene cloning: Prepare Insert DNA:

A

PCR amply and do restriction digest of DNA fragment to be cloned

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15
Q

What are the steps for gene cloning?

A
  1. Prepare Insert DNA
  2. Prepare Plasmid DNA
  3. Clean up
  4. Transform
  5. Plate
  6. Pick colonies, check by PCR
  7. Grow, isolate
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16
Q

Steps for gene cloning: Prepare Plasmid DNA:

A

purify in quantity, do restriction digest

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17
Q

Steps for gene cloning: clean up:

A

both insert and plasmid DNA (gel/column purify), mix plasmid and insert in varying ratios, ligate

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18
Q

Steps for gene cloning: transform:

A

competent cells of E. coli

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19
Q

Steps for gene cloning: plate:

A

onto media with selectable marker (e.g. ampicilin)

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20
Q

Steps for gene cloning: pick colonies, check by PCR:

A

for presence of insert DNA

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21
Q

Steps for gene cloning: grow:

A

in large culture, isolate cloned plasmid

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22
Q

substrate for Beta-galactosidase

A

X-Gal

23
Q

molecular mimic for allolactose

A

IPTG

24
Q

any DNA made from an RNA template; lacks introns

A

cDNA (complementary DNA)

25
Q

makes ssDNA from RNA

A

reverse transcriptase

26
Q

developed by Kary Mullis in 1985

A

PCR

27
Q

Template DNA, primers, dNTPs, thermostable DNA polymerase

A

PCR

28
Q

technique to measure mRNA; compare PCR gel product relative to known standard (e.g. actin); very sensitive, semi-quantitative

A

RT-PCR (reverse transcriptase PCR)

29
Q

Regular PCR is a ____ method.

A

semi-quantitative

30
Q

a quanitative measure of mRNA

A

real-time PCR

31
Q

modified thermocycler to detect fluorescence signals from tagged primers

A

real-time PCR

32
Q

vary standard and unknown mRNA template amounts

A

real-time PCR

33
Q

a method to detect a particular DNA sequence within a mixture of many DNA fragments

A

Southern blots

34
Q

developed by Edwin Southern in 1975

A

Southern blots

35
Q

What are the multiple applications of southern blotting?

A
  1. Determine copy number of a given gene in a genome
  2. Detect small gene deletions that cannot be detected by light microscopy
  3. Identify members of a gene family
  4. Identify homologous genes among different species
  5. Identify transgenes within a transgenic organism
36
Q

isotope or fluorescent label DNA fragment

A

DNA probe

37
Q

Stringency control of binding:

A

Use high (low) temperature and low (high) salt washes to require high (low) complementary binding of probe to fragments on membrane

38
Q

a technique used to detect mRNA species

A

northern blots

39
Q

developed in 1977 by George Stark

A

northern blots

40
Q

Determines whether a candidate gene is transcribed at all, or in a particular tissue

A

northern blots

41
Q

Similar procedure to southern blotting

A

northern blots

42
Q

Steps of northern blotting:

A
  1. isolate RNA
  2. Partially purify
  3. Separate on agarose gels with formaldehyde
  4. Transfer to nylon membrane
  5. Hybridise to labeled probe
  6. Image on film
43
Q

a technique used to detect the presence of a protein

A

western blots

44
Q

developed in 1979 by Harry Towbin

A

western blots

45
Q

What are the steps of western blotting?

A
  1. isolate proteins
  2. denature with SDs
  3. separate on polyacrylamide gels
  4. electrophorese transfer to a PVDF membrane
  5. block non-specific sites with NFDM
  6. hyrbridise to a 1 degree antibody
  7. hybridise to a 2 degree antibody with conjugated enzyme
  8. do chemiluminescence reaction and image on film
46
Q

method of DNA sequencing found by Frederick Sanger in 1970’s

A

dideoxy method

47
Q

Uses DNA polymerase to copy a strand of DNA

A

dideoxy method

48
Q

DNA synthesis is terminated at specific nucleotides by using analogs that lack a 3’ hydroxyl group

A

chain termination

49
Q

4 reactions, each with a small amount of one labeled dideoxynucleotide

A

dideoxy method

50
Q

Most sequencing reactions use:

A

fluorescently labeled dideoxynucleotides

51
Q

Aspects of fluorescently labeled dideoxynucleotides:

A
  1. samples are electrophoresed through a polyacrylamide gel in a small capillary tube
  2. as each band comes off the bottom of the gel, the fluorescent dye is excited by a laser
  3. the fluorescence emission is recorded by the fluorescence detector
52
Q

a method to make specific and intentional changes to the DNA sequence of a gene

A

site-directed mutagenesis

53
Q

What are the aspects of site-directed mutagenesis?

A
  1. important information about abnormal genetic processes

2. study affect of an amino acid alteration on protein function

Genetics (18 decks)

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